An important aspect in the development of gene therapy is the development of vectors capable of introducing genetic material into the target cells. In order to be effective, those vectors are required to possess several features: they must be capable of accommodating large or multiple transgenes inclusive of gene regulation elements, yet remaining simple to manipulate, such as to enable the production thereof on a pharmaceutical scale. Moreover, it is essential that they be safe and of a low toxicity, though preserving the capability of introducing the transgenes in an efficient and selective manner into the target tissues. Finally, the vector should preferably be compatible with an appropriate retention, expression and regulation of the transgene into the target cell.
At present, the adenovirus-based viral vectors seem to be the ones most suitable for manipulations making them capable of meeting all of those requirements. To date they are considered the most effective system for the introduction of heterologous genes in mammalian cells, both in vivo and in cells cultivated in vitro (Hitt M. M. et al.1997. Advances in Pharmacol.40, 137-206).
This is due to some interesting characteristics of the Adenoviruses (Ad), which constitute a DNA virus family (the ones infecting human beings have been classified in 57 serotypes), characterized by an icosahedral capsid lacking an outer coat: they are highly infective but relatively innocuous, primarily infect epithelial cells but can also infect cells of other tissues, regardless of their being in active replication phase. Because of their high infectiousness in vitro of cell lines of human origin, they can easily be produced in large amounts. Moreover, it has been proved that human DNA inserts can be efficiently transferred into epithelial human cells through Ad infection (Horvitz, “Adenoviridae and their replication” in Virology, Field and Knipe, ed. Raven Press, NY; 1990; pages 1679-1740).
With regard to molecular biology of the virus, adenovirus, in particular the human one, has a linear double-stranded DNA genome, of about 36 kb, functionally subdivided into two regions, non-structural and structural. The first one includes regions encoding polypeptides expressed in the first stages of the infection, i.e., prior to the viral DNA replication (E regions), the second ones includes regions coding for polypeptides expressed in the subsequent stages of the viral cycle (L region). Following the infection of a competent cell, when the viral DNA reaches the nucleus, the first region to be transcribed is the E1a region coding for proteins involved in the transactivation of the other regions, both E and L, of the viral DNA. The subsequently transcribed E1b region encodes proteins regulating the RNA synthesis, both viral and of the host cell, and protecting the latter from the apoptotic effect otherwise exerted by E1a. Therefore, the E1a/E1b functions are essential for the viral replication.
The E2 region encodes proteins directly involved in the viral replication, like the viral DNA-polymerase, the pre-terminal protein and proteins binding to the viral DNA. The E3 region encodes proteins that are unnecessary for the viral replication in cultured cells, but are involved, in vivo, in the regulation of the antiviral immune response. Lastly, the E4 region contains groups of genes whose products reduce the gene expression of the host cell and increase the transcription of the E2 and L regions of the viral genome.
The L region of the viral genome essentially encodes proteins of structural type, or anyway involved in the assembling of the viral particles.
In the 40 years following the first isolation, following the characterization of Ad viruses, the relevant modifications that made them efficient carriers for the transfer of genic material have been progressively developed.
In particular the interventions on the Ad genome have been firstly carried out in order to                increase the capability of the viral genome to accept the insertion of heterologous genes;        eliminate intracellular toxicity deriving from the expression of adenovirus genes.Such interventions consist mainly in the provision of progressive deletions of viral regions, whose functions are provided in trans.        
In a first generation vectors (derived from human Ad serotypes 2 and 5), the deletion has involved the E1 region, making the virus defective for the capability of replication, unless the proteins produced by such transcriptional unit are provided in trans.
An increase of the size of the heterologous gene to be inserted and the restriction of the propagation of the recombinant viruses in cell lines that complement such defect because they constitutionally express genes of the viral E1 region, have been obtained.
However, the deletion of E1 is not sufficient per se to completely eliminate the expression of other genes of the E and L regions, and to prevent the viral DNA replication. It follows that in animals infected with those vectors the presence of viral antigens and the onset of immune responses aiming at the destruction of the infected cells are detected (Yang et al. Proc. Natl. Acad. Sci. 91:4407-4411; 1994). This leads to the loss of the gene of therapeutic interest and to the onset of inflammatory reactions. Moreover, the persistence of an immunological memory of these reactions can greatly diminish the effectiveness of a second administration of an adenoviral vector of this type (Kozarsky et al. J. Biol; Chem. 269:1-8; 1994).
Hence new, second generation, adenoviral vectors carrying different combinations of early gene deletion have been constructed to improve the safety profile of Adenoviral vectors. Vectors differently combining E1, E2, E3, and/or E4 deletions have been demonstrated to be less cytotoxic in vitro and more stable in mouse liver than the classic ΔE1 first generation vectors (Gao, G-P. 1996 J. Virol 70:8934-8943; Dedieu, J-F. 1997 J. Virol. 71:4626-4637; Gorziglia, M.I. 1996 J. Virol. 70:4173-4178; Amalfitano, A. 1998 J. Virol. 72:926-933). In vivo experiments demonstrated that such deletions effectively diminished, but not abolished, the toxicity.
In addition, vectors carrying additional deletions further increasing the capability of the viral genome to accept the insertion of heterologous genes have been produced (Englehardt et al. Proc. Natl. Acad. Sci. 91:6196-6200; 1994; Bett et al. Proc. Natl. Acad. Sci. 91:8802-8806; 1994; Yeh, P., et al. 1996 J. Virol. 70:559-565). However, the maximum capacity of a first generation adenoviral vector does not exceed 8 kb, whereas that of a ΔE1/E3/E4 vector reaches 11 kb and the foreign genes can be equally inserted in the region E1 or E3.
In this context, following the discovery that a minimal portion, about 600 bp, of the viral DNA is strictly necessary for the replication and the encapsidation of recombinant vectors, totally defective adenoviral vectors, and consequently totally depending on the presence of helper viruses for the replication and the assembling thereof in viral particles, have been developed.
Such kind of helper-dependent adenoviral vectors (ADHD) carry minimal Ad sequences containing the Signals sufficient for the replication and the encapsidation. All the other factors necessary for virion production are provided in trans by a helper virus. The helper genome is constructed in such a manner that its sequences containing encapsidation signals can be easily eliminated in vivo through the use of specific recombination systems like the cre/lox system (WO97/32481).
Accordingly, the relevant strategies for the preparation of helper-dependent Adenovirus virions (adenoHD) are essentially based on the use of three elements:                cell lines transformed so as to make them capable of expressing the genes encoding the group of adenovirus E1 proteins, and a recombinase, usually “cre”;        a helper adenovirus wherein the E1 region is deleted and wherein the viral DNA sequences required for the encapsidation thereof inside the mature virion are flanked by recombination sites acting as substrates for the recombinase (“loxP” in case of “cre”);        the adenoHD vector carrying the transgene of interest.        
Although representing a remarkable improvement over the strategies utilising first generation vectors, this strategy presents some problems. For a production on a pharmaceutical scale, the requirement of controlling three independent components (the cell line, the helper and the transgenic vector) entails difficulties that are hard to overcome and unacceptable production costs.
Firstly, the cre-type recombinases catalyze both the deletion and the insertion of the DNA regions flanked by the loxP sites. The excision reaction is normally up to 20-fold more efficient than the opposite one, however a complete removal of the helper from the viral production can never be obtained. This is unacceptable especially in the pharmaceutical practice, and helper contamination of preparations of therapeutical use is a really serious problem.
A second limitation of this type of strategy is due to the difficulty of optimising the quantitative ratios between helper virus and helper-dependent virus during the amplification process. Hence, the vector amplification seldom approaches the efficiency of first generation vectors, and more often yields lesser to the extent of one order of magnitude are obtained. As in the previous case, this problem is dramatic when a production on an industrial scale is required, the costs becoming practically prohibitive.
In order to solve the problem deriving from the helper virus contamination and to reduce the number of components, an alternative strategy known in the art is that of engineering cell lines to make them capable of expressing all the factors necessary for the encapsidation of the defective virus, eliminating hence the helper virus.
In prior art there are several descriptions of cells expressing one or more viral proteins (see for instance WO98/13499). Such cell lines can be used for the production of adenoviral vectors defective of the complementary viral proteins. However, according to this strategy, it is extremely difficult to satisfactorily produce the exact co-ordination of the events between the viral DNA replication and the expression of the structural proteins that in the natural infection lead to the massive production of viral particles typical of the lytic phase. Accordingly, adopting these strategies, the viral cell cycle cannot be mimicked. In absence of this, only a limited yielding capacity may be achieved.